48 well culture plate

The SWFs from D580 hens were transferred to the DMEM high glucose (Hyclone, Tauranga, New Zealand) supplemented with 5% fetal calf serum (FCS; Hyclone, Utah), 100 IU/ml penicillin (Beyotime Biotechnology, Shanghai, China), 100 mg/ml streptomycin (Hyclone, Fremont, CA, United States), 2 mM glutamine and insulin-transferrin-selenium mixture (ITS: 10 mg/ml insulin, 5 mg/ml transferrin and 30 nM selenite). The follicles were cultured in 48-well culture plates (Corning Inc., Corning) with the medium at 38.5°C with 5% CO₂ for 72 h (Zhou et al., 2021 (link)). The medium was renewed every 24 h. The cultured SWFs from D580 hens were treated with 5 μg/ml (8.5 μM) GSPs (Liu X. T. et al., 2018 (link)) or/and 10 mM 3-methyladenine (3-MA).

hPBMCs, CD4⁺, or CD3⁺ T lymphocytes (5 × 10⁵ cells/well) were treated with phytohemagglutinin (PHA, 12.5 ug/ml, Sigma Aldrich, St. Louis, MO, USA) to stimulate T lymphocyte proliferation in the absence or presence of PGRN and incubated in 48-well culture plates (Corning, NY, USA) for 5 days. EdU (5-ethynyl-2-deoxyuridine, 10 μM, Invitrogen, Carlsbad, CA, USA) was added to the culture plates 18 hr before harvest. Based on a click reaction, EdU were reacted with azide, which is coupled to Alexa Flour 488 (Invitrogen, Carlsbad, CA, USA) dye. After harvesting the cells, the extent of cell proliferation was measured by flow cytometry (Beckman Coulter, Miami, FL, USA). Data were analyzed with FlowJo software (FlowJo, Ashland, OR, USA). Human TGF-β RI Kinase Inhibitor VI (SB431542, Selleckchem, Houston, TX, USA) or Treg inhibitor (Peptide P60, Abbiotec, San Diego, CA, USA) was used to block the formation of iTregs, and recombinant human TGF-β 1 protein (R&D System, Minneapolis, MN, USA) was used to induce the formation of iTregs.

The SWFs and ASWFs from high-laying (aged 280 ± 20 days) hens were transferred to the complete medium of DMEM high glucose (Hyclone, Tauranga, New Zealand) supplemented with 5% fetal calf serum (FCS; Hyclone, UT, USA). The follicles were cultured in 48-well culture plates (Corning Inc., Corning, NY, USA) at 38.5 °C and 5% CO₂ atmosphere for 72 h. ASWFs were treated with FSH (0.1 IU/mL) for 72 h [9 (link)].

The human keratinocytes (HaCaTs), human umbilical vein ECs (HUVECs), and human dermal fibroblasts (HDFs) were purchased from the cell bank, Chinese Academy of Sciences. To evaluate the cell proliferation on the PT-P, PT-P/GM, and 3D-PT-P/GM scaffolds, the nanofibrous scaffolds were sheared into round pieces with a diameter of 10 mm and sterilized with 75% ethanol for 15 min for twice, then washed with sterilized PBS thrice. The HUVECs, HDFs, and HaCaTs were seeded on the surfaces of scaffolds (PT-P, PT-P/GM, and 3D-PT-P/GM) with a density of 6 × 10³ cells per well in 48-well culture plates (Corning, USA) respectively and maintained at 37 °C under a humidified atmosphere with 5% CO₂. The viability of cells was measured by cell counting kit-8 (Kumamoto, Japan). The absorbance value of samples was measured at 450 nm using an enzyme-linked immunosorbent assay plate reader (Molecullar Devices).

Purified primary human monocytes from peripheral blood were obtained from the Human Immunology Core at the University of Pennsylvania (Philadelphia, PA, USA). The Human Immunology Core has the Institutional Review Board approval for blood collection from healthy donors. Freshly isolated monocytes were plated in 48-well culture plates (Corning CELLBIND Surface) at a density of 2.5 × 10⁵ cells/well in DMEM containing 10% fetal calf serum (FCS), 1% non-essential amino acid, 1% L-glutamine, and 1% penicillin–streptomycin solution at 37°C with 5% CO₂. Monocyte-derived macrophages refer to 7-day-cultured monocytes in vitro. HIV-infected U1 and OM10.1 cell lines were provided by the AIDS Reagent Program, National Institutes of Health. U1 is a cloned cell line derived by limiting dilution cloning of U937 cells surviving an acute infection with HIV (LAV-1 strain); each cell has two copies of integrated HIV proviral DNA (17 (link), 18 (link)). OM10.1 cells were cloned from HL-60 promyelocyte cells that endured an acute HIV infection. Each cell contains a single integrated provirus (19 (link), 20 (link)). U1 and OM10.1 cells were cultured in RPMI1640 supplemented with 10% FCS, 1% L-glutamine, and 1% penicillin–streptomycin.

To evaluate the cell attachment and infiltration on those scaffolds, the nanofibrous scaffolds were sterilized similarly to cell proliferation assay. HUVECs were seeded on the surfaces of PT-P, PT-P/GM, and 3D-PT-P/GM with a density of 2 × 10⁴ cells per well in 48-well culture plates (Corning, USA) and maintained at 37 °C under a humidified atmosphere with 5% CO₂ for 24 h. Then, the cells were fixed with 4% (w/v) paraformaldehyde for 15 min. Finally, Cells were then permeabilized with 0.1% Triton-X100 for 10 min and incubated with FITC-phalloidin at 37 °C for 1 h. Cell attachment and infiltration could be observed by laser scanning confocal microscope.